1. Field of the Invention
The present invention is directed to peptides that, in association with Class I MHC molecules, form epitopes recognized by cytotoxic T-cells specific for human melanoma.
2. Description of the Background Art
Melanoma affects 30,000 new patients per year in the United States. It is a cancer manifested by the unabated proliferation of melanocytes. Eighty percent of melanoma patients are diagnosed during their productive years between the ages of 25 and 65. The incidence of melanoma is rapidly increasing, in 1935 the lifetime risk of developing melanoma was 1:1,500 individuals, at present, the risk has risen to 1:105. It is believed that by the year 2000 the risk of developing melanoma will increase to about 1:70 to 1:90. Early diagnosis and treatment of this disease is crucial. Once a primary tumor becomes metastatic the disease is almost uniformly fatal.
Cytotoxic lymphocyte (CTL) response has been shown to be an important host defense against malignant cells, Rock et al. J. Immunol., (1993), 150:1244. Several studies have shown the presence of shared epitopes located on melanoma cells. CTLs respond to these epitopes, however, until now the epitopes were structurally undefined. Anti-tumor CTL may be generated in vitro by the restimulation of lymphocytes isolated from melanoma patients using autologous tumor cells, Slingluff et al., Arch. Surg., (1987), 122:1407. Adoptive transfer of tumor stimulated CTL has been associated with some tumor regressions, Rosenberg et al., N. Eng. J. Med., (1988), 319:1676. However, cure is rarely mediated by this approach. Alternate approaches to augmenting the T-cell response to melanoma have been the use of tumor vaccines, also known as specific active immunotherapy.
Attempts to maintain competent levels of immunity against emergent tumor cell clones using melanoma antigen have done little to affect the natural course of the disease. This may be due, in part, to the non-specific nature of antigen used. Thus, there is a need to provide specific tumor antigens or haptens for the purpose of enhancing the immune response to tumors cells in order to effectively vaccinate against tumor cell growth.
It is believed that understanding the cellular immune response to melanoma may be important in defining new therapies; however, optimizing those therapies will depend on identification of the specific epitopes recognized by those CTL.
Lymphocytes isolated from patients having melanoma, when stimulated in vitro with recombinant interleukin-2 (rIL-2) and autologous melanoma cells, develop a melanoma specific cytotoxic response, Vose et al., Nature, (1982), 296:359; Knuth et al., Proc. Natl. Acad. USA, (1984), 81:3511; Slingluff et al., Arch. Surg., (1987), 122:1407; Darrow et al., Cancer, (1988), 62:84; Slingluff et al., J. Natl. Cancer Inst., (1988), 80:1016; Slingluff et al., Ann. Surg., (1989), 210:194; Muul et al., J. Immunol., (1987), 138:989; Anichini et al., Int. J. Cancer, (1985), 35:683. Melanoma specific effector lymphocytes are, by the majority, CD8+ cytotoxic T lymphocytes (CTL) that are restricted by class I Major Histocompatibility Complex (MHC) molecules, Vose et al; Slingluff et al (1988), supra, Hersey et al., Cancer Immunol. Immunother., (1986), 22:15. These characteristics are present whether CTL have been generated from peripheral blood lymphocytes (PBL), Vose et al.; Slingluff et al. (1989), supra; Van den Eynde et al., Int. J. Cancer, (1989), 44:634, lymph node cells, or tumor infiltrating lymphocytes. Evidence that the CTL response to human melanoma is restricted by class I MHC molecules includes demonstration of cross-reactivity for allogenic melanoma cells that share a restricting class I MHC molecule with the autologous tumor. The HLA-A2 molecule and its variants, of which HLA-A2.1 is by far the most common, is an effective restricting element for the melanoma-specific CTL response. Additionally, melanoma-specific HLA-restricted CTL lyse the majority of A2+ melanomas tested, Darrow et al., J. Immunol., (1989), 142:3329; Wolfel et al., J. Exp. Med., (1989), 170:797; Hom et al., J. Immunother., (1991), 3:153. By demonstrating lysis of A2-melanomas transfected with the A2.1 gene, it has been shown that these transfected melanomas can present the epitopes recognized by A2-restricted melanoma-specific CTL, Kawakami et al., J. Immunol., (1992), 148:638. These results suggest that these CTL recognize A2-restricted epitopes that are shared by the majority of melanomas, although very little is known about the number and identity of their epitopes.
Epitopes for CD8+ CTL are believed to be short, usually 9-residue peptides that bind to a cleft on the surface of the class I MHC molecule, Udaka et al., Cell, (1992), 69:989; VanBleek et al., Nature, (1990), 348:213; Falk et al., J. Exp. Med., (1991), 174:425. These peptides, generated from proteolysis of endogenous proteins in the cytosol, are transported to the endoplasmic reticulum, where they become associated with newly synthesized class I MHC molecules. They are then transported to the cell surface, Elliott et al., Nature, (1990), 3348:195. Because of the complexity of the peptide mixture associated with class I MHC molecules, Hunt et al., Science, (1992), 255:1261, the definition of individual peptides that comprise specific CTL epitopes has proved extremely difficult. One approach has been to identify the genomic sequence coding for a CTL epitope using Ag loss variants of a melanoma line. The gene encoding a single HLA-A1-restricted melanoma epitope has been isolated by this method, Van der Bruggen et al., Science, (1991), 254:1643. However, only a subset of HLA-A1+ melanomas express this gene. Thus peptides identified using this method would not provide an effective therapy for a majority of melanoma patients.
Tyrosinase (monophenol, 3,4-dihydroxyphenylalanine: oxidoreductase, E.C. 1.14.18.1) has been shown to be specifically expressed by melanocytes and melanoma cells, Brichard et al., supra. The enzyme catalyses the synthesis of dihydroxyphenylalanine (DOPA) in the first two steps of melanin biosynthesis. Autologous CTL recognize an antigen on tumors apparently derived from the tyrosinase molecule. However, this antigen is not recognized by most CTL lines tested.
An alternate approach toward characterization of CTL epitopes is to identify them directly. CTL epitopes have been reconstituted in vitro by allowing exogenous peptides to bind to MHC molecules on the cell surface of target cells, Townsend et al., Annu. Rev. Immunol., (1989), 7:601. To use this approach for the identification of clinically important melanoma-specific epitopes, it is necessary first to demonstrate that peptides comprising these epitopes can be extracted from melanoma cells and that these epitopes are shared by different melanoma lines.
In the present invention, HLA associated peptides have been extracted, isolated and identified from different melanoma lines. These peptides can be used to reconstitute epitopes for HLA-A2.1-restricted melanoma-specific CTL. These peptides and the resulting CTL may be useful for the in vivo immunotherapeutic treatment of melanoma.
The present invention relates to immunogens which are capable of eliciting a melanoma-specific cytotoxic lymphocyte response in at least some individuals, which response is directed to peptide epitopes carried by those immunogens, and to the use of those immunogens in active specific immunotherapy and immunoprophylaxis against melanoma.
The instant disclosure identifies and synthesizes peptide residues initially isolated from a melanoma cell line. The peptides are capable of reconstituting an epitope recognized by tumor specific CTL. Some of the sequences are homologous with proteins identified as pMEL17, tyrosinase and cofilin. The most active of these sequences have been recognized from 5 out of 5 melanoma patients tested. Fundamentally, the present invention provides for the treatment of melanoma patients using synthetic peptides that reconstitute epitopes for melanoma specific CTL. In another embodiment the peptides are used as vaccines for imparting immunity. Alternatively, in one embodiment the peptides may be used to bind to antigen presenting cells in a method for providing specific antigenic stimulation of CTL. The instant invention provides CTL cell lines capable of recognizing reconstituted HLA-A2.1 epitopes and their use in methods of adoptive immunotherapy. The invention additionally provides for genes encoding for peptides capable of reconstituting epitopes recognized by tumor specific CTL and their use as vaccines in the prevention and management of melanoma.
To identify the active peptides a splitter was devised.
In addition to the potential application of antigenic peptides as the basis of a tumor vaccine in man, there are at least three other utilities: (1) adoptive transfer of tumor-specific CTL generated by stimulation of CTL with antigenic peptides in vitro, and (2) use of peptide bound to target cells (eg T2) for evaluation of the immune response of patients, and (3) use, especially of peptides 946L, and 946I as laboratory reagents for evaluation of peptide/MHC binding affinity.